CN111218011B - Polyethylene glycol-based hydrogel and preparation method and application thereof - Google Patents
Polyethylene glycol-based hydrogel and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a polyethylene glycol-based hydrogel and a preparation method and application thereof, belonging to the technical field of gel material preparation. The preparation method comprises the following steps: synthesizing four-arm PEGNB by using four-arm polyethylene glycol amino and norbornene as raw materials under the auxiliary action of a condensing agent HATU and an organic base DIPEA; then PEGNB, polyethylene glycol dithiol and a photoinitiator I2959 initiate click chemistry reaction under the action of ultraviolet to form gel, and then the PEG-based hydrogel is modified by arginyl-glycyl-aspartic acid tripeptide on the basis of gel formation. The preparation technology of the invention has simple steps, and the copolymer is formed by photocatalysis, thereby avoiding the problem that the catalyst is difficult to remove. The invention can realize the controllability of the hardness and the mechanical property of the hydrogel by changing the mixture ratio of substances in the hydrogel precursor formula. The invention has short gelling time and good gel stability, and establishes a new in vitro research model for simulating in vivo extracellular matrix.
Description
Technical Field
The invention relates to the technical field of gel materials, in particular to a preparation method and application of polyethylene glycol-based hydrogel.
Background
Polyethylene glycol (PEG) is a water-soluble polymer compound, has many advantages such as low toxicity, no blood coagulation, and good biocompatibility, and is widely used in the fields of medicine, health, chemical industry, food, etc. An important application of PEG is in the preparation of hydrogels. Hydrogels are highly hydrophilic polymeric networks obtained by polymerizing and crosslinking one or more monomers using chemical initiation, ionizing radiation, or ultraviolet radiation. Typically, a small amount of crosslinking agent is added during the formation of the hydrogel. Hydrogels have a wide range of applications in the biomedical field. PEG-based hydrogels resemble living tissue more than other synthetic biomaterials.
Currently, many methods for preparing PEG-based hydrogels are known, such as diels-alder cycloaddition reaction, azide-alkyne reaction, oximation reaction, etc., but the following disadvantages generally exist:
(1) the preparation process is complex, the catalyst is difficult to remove, and other mixed substances are easy to generate;
(2) the hydrogel has poor gelling effect and poor mechanical property;
(3) easy to generate immunogenicity and influence the biocompatibility.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a preparation method and application of polyethylene glycol-based hydrogel, wherein the preparation method comprises the steps of taking four-arm polyethylene glycol amino and norbornene as raw materials, and taking a condensing agent HATU and an organic base DIPEA as auxiliary materials to synthesize four-arm PEGNB; then PEGNB, polyethylene glycol dithiol and a photoinitiator I2959 initiate click chemistry reaction under the action of ultraviolet to form gel, and then the gel is modified by arginyl-glycyl-aspartic acid tripeptide on the basis of gelling to obtain the hydrogel material. The specific technical scheme is as follows:
a preparation method of polyethylene glycol-based hydrogel comprises the following steps:
(1) synthesis of four-armed PEGNB
Taking a four-arm polyethylene glycol amino solution and a norbornene solution as raw materials, taking a condensing agent HATU and an organic base DIPEA as auxiliary materials, reacting under inert and sealed conditions to obtain a PEGNB solution, then adding the PEGNB solution into glacial ethyl ether, stirring, performing suction filtration to obtain a wax, and purifying the wax to obtain the four-arm PEGNB;
(2) preparation of polyethylene glycol-based hydrogel
Mixing the PEGNB prepared in the step (1) with polyethylene glycol dithiol, I2959 working solution and deionized water to obtain hydrogel precursor solution, dropwise adding the hydrogel precursor solution to a lower glass slide treated by TMPTMA, covering an upper glass slide treated by a silane coupling agent, irradiating the glass slide to form glue under an ultraviolet condition, taking down the upper glass slide, dropwise adding a mixed solution of the I2959 working solution and RGD working solution to the surface of the glued gel, covering the upper glass slide again, and irradiating the glass slide under the ultraviolet condition to obtain the polyethylene glycol-based hydrogel.
In order to obtain the PEGNB hydrogel with different elastic moduli, the proportion of each glue-making component is adjusted, and the general PEGNB mass percentage concentration is 6-12 wt%, the polyethylene glycol dithiol mass percentage concentration is 1.1-4.4 wt%, the I2959 mass percentage concentration is 0.5-5 wt%, and the deionized water mass percentage concentration is 80-92 wt%.
The four-arm polyethylene glycol norbornene hydrogel is obtained by selecting raw materials and combining a thiol-ene-click chemical reaction principle, can be used for simulating an extracellular matrix of a cell membrane, is adjustable in hardness, is suitable for research under a healthy or pathological state, is wide in application, simple in whole preparation process, short in gelling time, good in stability of the obtained colloid, and well improved in biocompatibility. The two stages of the preparation process of the present invention are further illustrated below in connection with the selection of starting materials for the present invention.
The first stage is as follows: synthesis of a four-arm polyethylene glycol norbornene (PEGNB). The invention uses four-arm polyethylene glycol amino (4 arm-PEG-NH)2) Is a main raw material due to amino (-NH) in the molecule2) Active groups capable of reacting with primary amine can be modified to form stable amido bonds with carboxyl (-COOH) and form amido bonds with active ester (-NHS) at the pH value of 7-8.5, and PEG groups can increase the solubility and stability of drugs, reduce the immunogenicity of polypeptides and proteins, inhibit the non-specific combination of charged molecules on the modified surface, and can also be modified by proteins, polypeptides and other small molecular materials. The invention uses Norbornene (Norbornene, abbreviated as NB) and 4arm-PEG-NH2Four-arm polyethylene glycol (PEGNB) was synthesized. The NB molecule contains a molecule with a double bond which can cause obvious ring tension and obvious reaction, so the invention utilizes the addition of norbornene into 4arm-PEG-NH2The polymer can obtain a material with good thermal stability, high optical transparency, low hygroscopicity and low dielectric constant, and is used as subsequent polyethylene glycol-based waterThe preparation of the gel lays an excellent performance foundation. Meanwhile, when synthesizing PEGNB, the invention also adds a condensing agent HATU and an organic base DIPEA to promote the reaction, wherein HATU is a reagent for peptide coupling reaction, can activate carboxyl in NB with the assistance of DIPEA, and reacts with 4arm-PEG-NH2Salifying the amino group, dehydrating to form an amide, and finally forming a four-arm PEGNB structure shown as a structural formula A or B. Wherein A is a structural formula and B is a molecular formula.
Since the reaction system needs to be carried out under anhydrous conditions, the present invention uses an organic solvent to dissolve the starting material sample.
And a second stage: the method comprises the steps of mixing synthesized PEGNB with I2959 and polyethylene glycol dithiol to obtain a hydrogel precursor, wherein I2959 is a high-efficiency non-yellowing ultraviolet initiator and is used for initiating an ultraviolet polymerization reaction of an unsaturated prepolymerization system, I2959 generates free radicals under the irradiation of ultraviolet light, and the free radicals initiate a click chemical reaction through sulfydryl of polyethylene glycol dithiol to react with double bonds on NB to form a network structure to form hydrogel. In addition, the hydrogel is modified by arginyl-glycyl-aspartic acid (RGD) based on gelling, and the arginyl-glycyl-aspartic acid tripeptide sequence is a tripeptide sequence consisting of arginine, glycine and aspartic acid, can be specifically combined with 11 types of integrins, can effectively promote the adhesion of cells to biological materials, is fixed on the surface energy of the hydrogel, and can well improve the biocompatibility of the hydrogel. In addition to the RGD improving the biocompatibility of the surface of the PEGNB hydrogel, other small peptides, polypeptides or proteins or other small molecules capable of promoting cell growth can be introduced into the surface of the PEGNB hydrogel to enhance the biocompatibility.
Further, in a preferred embodiment of the present invention, the step (1) comprises the following specific steps:
(1.1) dissolving 2g of four-arm polyethylene glycol amino in a sufficient amount of organic solvent to obtain a four-arm polyethylene glycol amino solution;
(1.2) weighing 2-3 g of HATU, adding 0.5-1 g of NB and a sufficient amount of organic solvent under an inert atmosphere, dissolving for 5-10 mins, adding 2-3 ml of DIPEA, and mixing until the HATU is completely dissolved to obtain a solution product;
(1.3) dropwise adding the solution product prepared in the step (12) into a four-arm polyethylene glycol amino solution, and reacting under inert and sealed conditions to obtain a PEGNB solution;
(1.4) the PEGNB solution was added to 200mL of glacial ethyl ether and stirred, and a wax was obtained by suction filtration and purified.
Further, in a preferred embodiment of the present invention, the specific process of the above purification is: and (3) carrying out vacuum drying on the wax-like substance for 4-6 h, dissolving the wax-like substance in deionized water, dialyzing the wax-like substance for 2-3 days by using the deionized water, changing the water for 2-3 times every day, and then pre-freezing and freeze-drying the wax-like substance to obtain the four-arm PEGNB.
Further, in a preferred embodiment of the present invention, the molecular weight of the four-arm peg amino group is 4000 to 6000.
The molecular weight of the four-arm polyethylene glycol amino is 4000-6000, and the density of the gel material is adjusted in the range, so that colloids with different hardness degrees are obtained.
Further, in a preferred embodiment of the present invention, in step (2): the mass percent concentration of the PEGNB is 6-12 wt%, the mass percent concentration of the polyethylene glycol dithiol is 1.1-4.4 wt%, the mass percent concentration of the I2959 is 0.5-5 wt%, the mass percent concentration of the deionized water is 80-92 wt%, and the dropping amount of the hydrogel precursor solution is 15-20 mL.
Further, in a preferred embodiment of the present invention, in step (2): the mass percentage concentration of the I2959 working solution is 0.05wt%, the mass percentage concentration of the RGD working solution is 0.05wt%, and the dropping amount of the mixed solution of the I2959 working solution and the RGD working solution is 15-20 mu L.
Further, in a preferred embodiment of the present invention, in step (2): the time of the ultraviolet irradiation for the two times is 15-25 min and 5-15 min respectively.
The polyethylene glycol-based hydrogel prepared by the preparation method has the following structural formula:
The polyethylene glycol-based hydrogel is applied to preparation of extracellular matrix simulation materials.
The invention has the following beneficial effects:
the preparation method has simple preparation technical steps, and the copolymer is formed through photocatalysis, so that the problem that the catalyst is difficult to remove is avoided, and other mixed substances are difficult to generate. The invention can realize the controllability of the hardness and the mechanical property of the hydrogel by changing the mixture ratio of substances in the hydrogel precursor formula. The invention has short gelling time and good gelling stability, and can achieve good gelling effect only by a very small amount.
Meanwhile, in order to reduce the potential defect of the hydrogel prepared by a chemical method on biocompatibility and reduce the immunogenicity of the gel, the invention uses arginyl-glycyl-aspartic acid tripeptide to modify the PEG-based hydrogel so as to improve the biocompatibility of the PEG-based hydrogel and has no cytotoxicity.
The PEG-based hydrogel prepared by the invention has adjustable hardness, and can be applied to in vitro simulation of extracellular matrix in healthy state and pathological condition; provides a stable in vitro model for researching the influence of the change of the elastic modulus of the extracellular matrix on the cell growth, applying the change to the drug screening by simulating pathological states, applying the change to the drug screening in the field of other biomedical materials and the like, and establishes a novel in vitro research model for simulating the in vivo extracellular matrix.
Drawings
FIG. 1 is a NMR spectrum of a polyethylene glycol-based hydrogel prepared according to an example of the present invention;
FIG. 2 is a photograph showing a stability test of a polyethylene glycol-based hydrogel prepared according to an example of the present invention;
FIG. 3 is a graph showing the results of measuring the elastic modulus of a polyethylene glycol-based hydrogel prepared according to an embodiment of the present invention;
FIG. 4 is a diagram of a cell experiment of a PEG-based hydrogel prepared according to an embodiment of the present invention;
FIG. 5 is a cell activity assay of polyethylene glycol-based hydrogels prepared according to embodiments of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used are conventional products available from commercial sources, not indicated by the manufacturer.
The raw materials adopted by the invention comprise:
four-arm polyethylene glycol amino: english name 4arm-PEG-NH2。
HATU: it is called 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, (2- (7-Azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate.
DIPEA: it is called N, N-Diisopropylethylamine and N, N-dissopropylethylamine.
NB: known as Norbornene, Norbornene.
DMF: is called N, N-dimethylformamide, N, N-Dimethylformamid.
DCM: known as Dichloromethane, dichromethane.
PEGNB: substituted polyethylene glycol norbornene.
PEGNB hydrogel: and is polyethylene glycol-based hydrogel.
Inert gases used in embodiments of the present invention include, but are not limited to, argon.
Organic solvents useful in embodiments of the present invention include, but are not limited to DMF and DCM.
The ethyl ether used in the embodiment of the invention is obtained by freezing the ethyl ether in advance.
Example 1:
the preparation method of the polyethylene glycol-based hydrogel comprises the following steps:
(1) synthesis of four-armed PEGNB
Taking a four-arm polyethylene glycol amino solution and an NB solution as raw materials, taking a condensing agent HATU and an organic base DIPEA as auxiliary materials, reacting under inert and sealed conditions to obtain a PEGNB solution, then adding the PEGNB solution into glacial ethyl ether, stirring, performing suction filtration to obtain a wax, and purifying the wax to obtain the four-arm PEGNB;
(2) preparation of polyethylene glycol-based hydrogel
Mixing the PEGNB prepared in the step (1) with polyethylene glycol dithiol, I2959 working solution and deionized water, wherein the mass percentage concentration of the PEGNB is 6 wt%, the mass percentage concentration of the polyethylene glycol dithiol is 1.1 wt%, the mass percentage concentration of the I2959 is 0.9 wt%, and the mass percentage concentration of the deionized water is 92wt% to obtain hydrogel precursor solution, dropwise adding the hydrogel precursor solution onto a lower glass slide treated by TMPTMA, covering an upper glass slide treated by a silane coupling agent, irradiating the upper glass slide into glue under an ultraviolet condition, then taking down the upper glass slide, dropwise adding a mixed solution of the I2959 working solution and RGD working solution onto the surface of the glue-formed gel, covering the upper glass slide again, and irradiating the glue under the ultraviolet condition to obtain the polyethylene glycol-based hydrogel.
Example 2:
the preparation method of the polyethylene glycol-based hydrogel comprises the following steps:
(1) synthesis of four-armed PEGNB
(11) 2g of four-arm polyethylene glycol amino with the molecular weight of 4000-6000 is dissolved in sufficient organic solvent to obtain a four-arm polyethylene glycol amino solution;
(12) weighing 2g of HATU, adding 0.5g of NB and a sufficient amount of organic solvent under an inert atmosphere, dissolving for 5mins, adding 2ml of DIPEA, and mixing until the HATU is completely dissolved to obtain a solution product;
(13) dropwise adding the solution product prepared in the step (12) into a four-arm polyethylene glycol amino solution, and reacting under inert and sealed conditions to obtain a PEGNB solution;
(14) the PEGNB solution was added to 200mL of glacial ethyl ether and stirred, and the wax was purified by suction filtration.
The specific process of the purification is as follows: the wax was vacuum dried for 4h and dissolved in deionized water, then dialyzed against deionized water for 2 days, with 2 water changes per day, and then prefreezed and freeze dried to obtain the four armed PEGNB.
(2) Preparation of polyethylene glycol-based hydrogel
Mixing the PEGNB prepared in the step (1) with polyethylene glycol dithiol, I2959 working solution and deionized water, wherein the mass percentage concentration of the PEGNB is 10 wt%, the mass percentage concentration of the polyethylene glycol dithiol is 1.8 wt%, the mass percentage concentration of the I2959 is 1 wt%, and the mass percentage concentration of the deionized water is 87.2 wt% to obtain hydrogel precursor solution, dripping 15mL of the hydrogel precursor solution onto a lower glass slide treated by TMPTMA, covering an upper glass slide treated by a silane coupling agent, irradiating for 15min under an ultraviolet condition to form gel, taking down the upper glass slide, dripping 15 mu L of mixed solution of the I2959 working solution with the concentration of 0.05wt% and the RGD working solution with the concentration of 0.05wt% onto the gel surface formed by the gel, covering the upper glass slide again, and irradiating for 5min under the ultraviolet condition to obtain the polyethylene glycol-based hydrogel.
Example 3:
the preparation method of the polyethylene glycol-based hydrogel comprises the following steps:
(1) synthesis of four-armed PEGNB
(11) 2g of four-arm polyethylene glycol amino with the molecular weight of 4000-6000 is dissolved in sufficient organic solvent to obtain a four-arm polyethylene glycol amino solution;
(12) weighing 3g of HATU, adding 1g of NB and a sufficient amount of organic solvent under an inert atmosphere, dissolving for 10mins, adding 3ml of DIPEA, and mixing until the HATU is completely dissolved to obtain a solution product;
(13) dropwise adding the solution product prepared in the step (12) into a four-arm polyethylene glycol amino solution, and reacting under inert and sealed conditions to obtain a PEGNB solution;
(14) the PEGNB solution was added to 200mL of glacial ethyl ether and stirred, and the wax was purified by suction filtration.
The specific process of the purification is as follows: the wax was vacuum dried for 6h and dissolved in deionized water, then dialyzed against deionized water for 3 days, with 3 water changes per day, and then prefreezed and freeze dried to obtain the four armed PEGNB.
(2) Preparation of polyethylene glycol-based hydrogel
Mixing the PEGNB prepared in the step (1) with polyethylene glycol dithiol, I2959 working solution and deionized water, wherein the mass percentage concentration of the PEGNB is 7.9 wt%, the mass percentage concentration of the polyethylene glycol dithiol is 1.5 wt%, the mass percentage concentration of the I2959 is 0.1 wt%, and the mass percentage concentration of the deionized water is 90.5 wt% to obtain hydrogel precursor solution, dripping 20mL of the hydrogel precursor solution onto a lower glass sheet treated by TMPTMA, covering an upper glass sheet treated by a silane coupling agent, irradiating for 25min under an ultraviolet condition to form gel, taking down the upper glass sheet, dripping 20 mu L of mixed solution of the I2959 working solution with the concentration of 0.05wt% and the RGD working solution with the concentration of 0.05wt% onto the gel surface, covering the upper glass sheet again, and irradiating for 15min under the ultraviolet condition to obtain the polyethylene glycol-based hydrogel.
Example 4:
the preparation method of the polyethylene glycol-based hydrogel comprises the following steps:
(1) synthesis of four-armed PEGNB
(11) 2g of four-arm polyethylene glycol amino with the molecular weight of 4000-6000 is dissolved in sufficient organic solvent to obtain a four-arm polyethylene glycol amino solution;
(12) weighing 2.5g of HATU, adding 0.8g of NB and enough organic solvent under an inert atmosphere, dissolving for 8mins, adding 2.5ml of DIPEA, and mixing until the HATU is completely dissolved to obtain a solution product;
(13) dropwise adding the solution product prepared in the step (12) into a four-arm polyethylene glycol amino solution, and reacting under inert and sealed conditions to obtain a PEGNB solution;
(14) the PEGNB solution was added to 200mL of glacial ethyl ether and stirred, and the wax was purified by suction filtration.
The specific process of the purification is as follows: the wax is dried in vacuum for 5h and then dissolved in deionized water, then dialyzed against deionized water for 2.5 days, changed in water 3 times a day, prefreezed and freeze-dried to obtain the four-armed PEGNB.
(2) Preparation of polyethylene glycol-based hydrogel
Mixing the PEGNB prepared in the step (1) with polyethylene glycol dithiol, I2959 working solution and deionized water, wherein the mass percentage concentration of the PEGNB is 12wt%, the mass percentage concentration of the polyethylene glycol dithiol is 4.4wt%, the mass percentage concentration of the I2959 is 5wt%, and the mass percentage concentration of the deionized water is 78.6 wt% to obtain hydrogel precursor solution, dripping 18mL of the hydrogel precursor solution onto a lower glass slide treated by TMPTMA, covering an upper glass slide treated by a silane coupling agent, irradiating for 20min under an ultraviolet condition to form gel, taking down the upper glass slide, dripping 18 mu L of mixed solution of the I2959 working solution with the concentration of 0.05wt% and the RGD working solution with the concentration of 0.05wt% onto the gel surface formed by the gel, covering the upper glass slide again, and irradiating for 10min under the ultraviolet condition to obtain the polyethylene glycol-based hydrogel.
Test examples
1. Nuclear magnetic resonance spectroscopy
Nmr spectroscopy was performed on the PEGNB prepared in the examples of the present invention. Analysis by the MestReNova software showed that the peak for norbornene was normalized to 5.73, indicating that a total of 6 hydrogens had been grafted, while the PEGNB had a total of 8 hydrogens, indicating that the degree of grafting was 6/8-75%, as shown in figure 1. The grafting rate of the PEGNB compound prepared in the embodiment 3 of the invention is more than 90%.
2. Stability test
Referring to fig. 2, fig. 2 (before soaking) shows the gnb hydrogel in an un-soaked state, and fig. 2 (after soaking) shows the gnb hydrogel after soaking in the PBS solution for 15 days. As can be seen from fig. 2, the PEGNB hydrogel is prepared on a circular slide, the hydrogel is colorless and transparent, the surface of the colloid is flat, the edge of the colloid has no protrusion, small bubbles are generated between the slide and the gel, but the gel is not peeled off from the slide; after soaking in the PBS solution at 4 ℃ for 15 days, the PBS solution is removed, the thickness of the PEGNB hydrogel is slightly increased, the edge of the gel has slight wavy fluctuation due to the swelling effect, but the surface of the hydrogel is still flat without damage signs, and the gel is not peeled from the glass slide, which shows that the PEGNB hydrogel prepared by the invention can still keep stable gel property after being soaked for a long time and has longer service life.
3. Elasticity test
Referring to fig. 3, in the preparation of hydrogel, two representative soft and hard hydrogels were prepared by adjusting the molar ratio of polyethylene glycol dithiol to PEGNB. The molar ratio of polyethylene glycol dithiol to PEGNB is 0.5 and 0.7 respectively, which respectively represents the extracellular matrix of the soft and hard water gel in a simulated normal state and the extracellular matrix in a pathological state.
4. Biocompatibility test
The good biocompatibility of the PEGNB hydrogel is verified through cell experiments. The cells cultured by the blank pore plate are used as a control group, the bovine pulmonary artery endothelial cells are inoculated on the surfaces of the blank pore plate, the softer PEGNB hydrogel and the harder PEGNB hydrogel, the cells are cultured for 4 days, and microscopic observation is carried out after nuclear staining and cytoplasmic staining, so that the experimental result shows that the cells have strong refractivity, full cytoplasm and clear and good nuclear cytoplasm growth, as shown in figure 4. In addition, the biocompatibility of the PEGNB hydrogel was quantitatively determined by CCK-8 cell viability assay, and the results are shown in FIG. 5. The number of the growing cells on the PEGNB hard water gel and the pore plate has no significant difference, and the difference with the number of the growing cells on the surface of the soft water gel is significant. According to the cell experiments, the growth states of the cells on the surfaces with different hardness are different.
In conclusion, the preparation method disclosed by the invention can be used for obtaining hydrogels with different hardness degrees, namely adjusting the hardness of the extracellular matrix in the elastic modulus simulation pathological state, so that the application research of the PEGNB hydrogel in more medical application fields can be developed.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (7)
1. The preparation method of the polyethylene glycol-based hydrogel is characterized by comprising the following steps:
(1) synthesis of four-armed PEGNB
Reacting a four-arm polyethylene glycol amino solution and a norbornene solution serving as raw materials under an inert and sealed condition with a condensing agent HATU and an organic base DIPEA serving as auxiliaries to obtain a PEGNB solution, adding the PEGNB solution into glacial ethyl ether, stirring, performing suction filtration to obtain a wax, and purifying the wax to obtain the four-arm PEGNB;
(2) preparation of polyethylene glycol-based hydrogel
Mixing the PEGNB prepared in the step (1) with polyethylene glycol dithiol, I2959 working solution and deionized water to obtain hydrogel precursor solution, dropwise adding the hydrogel precursor solution onto a lower glass slide treated by TMPTMA, covering an upper glass slide treated by a silane coupling agent, irradiating under an ultraviolet condition to form gel, taking down the upper glass slide, dropwise adding a mixed solution of the I2959 working solution and RGD working solution onto the surface of the gel formed with the gel, covering the upper glass slide again, and irradiating under the ultraviolet condition to prepare polyethylene glycol-based hydrogel;
the RGD working solution is arginyl-glycyl-aspartic acid tripeptide; the molecular weight of the four-arm polyethylene glycol amino is 4000-6000; the time of the ultraviolet irradiation for the two times is 15-25 min and 5-15 min respectively.
2. The method for preparing the polyethylene glycol-based hydrogel according to claim 1, wherein the step (1) comprises the following specific steps:
(1.1) dissolving 2g of four-arm polyethylene glycol amino in enough organic solvent to obtain a four-arm polyethylene glycol amino solution;
(1.2) weighing 2-3 g of HATU, adding 0.5-1 g of norbornene and enough organic solvent in an inert atmosphere, dissolving for 5-10 mins, adding 2-3 ml of DIPEA, and mixing until the HATU is completely dissolved to obtain a solution product;
(1.3) dropwise adding the solution product prepared in the step (1.2) into a four-arm polyethylene glycol amino solution, and reacting under inert and sealed conditions to obtain a PEGNB solution;
(1.4) the PEGNB solution is added to 200mL of glacial ethyl ether and stirred to give a wax which is purified by suction filtration.
3. The method for preparing the polyethylene glycol-based hydrogel according to claim 2, wherein the purification comprises the following steps: and (3) drying the wax-like substance in vacuum for 4-6 h, dissolving the wax-like substance in deionized water, dialyzing the wax-like substance for 2-3 days by using the deionized water, changing the water for 2-3 times every day, and pre-freezing and freeze-drying the water to obtain the four-arm PEGNB.
4. The method for preparing a polyethylene glycol-based hydrogel according to any one of claims 1 to 3, wherein in the step (2): the mass percent concentration of the PEGNB is 6-12 wt%, the mass percent concentration of the polyethylene glycol dithiol is 1.1-4.4 wt%, the final mass percent concentration of I2959 is 0.5-5 wt%, the mass percent concentration of the deionized water is 80-92 wt%, and the dropping amount of the hydrogel precursor solution is 15-20 mL.
5. The method for preparing a polyethylene glycol-based hydrogel according to claim 4, wherein in the step (2): the mass percentage concentration of the I2959 working solution added for the second time is 0.05wt%, the mass percentage concentration of the RGD working solution is 0.05wt%, and the dripping amount of the mixed solution of the I2959 working solution and the RGD working solution is 15-20 mu L.
6. The polyethylene glycol-based hydrogel prepared by the method of any one of claims 1-5, wherein the four-armed PEGNB has the following structural formula:
7. Use of the polyethylene glycol-based hydrogel of claim 6 for the preparation of an extracellular matrix mimetic material.
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